RESUMEN
PURPOSE: Successful management of dysfunctional voiding in children hinges on retraining inappropriate pelvic floor muscle recruitment. Recently dynamic pelvic floor muscle activity was visualized in adults using transabdominal ultrasound. We evaluated transabdominal ultrasound for visualizing and measuring pelvic floor muscle activity in normative children. MATERIALS AND METHODS: A total of 21 volunteers, including 10 boys and 11 girls 7 to 16 years old (mean age 11.6) who were free of bladder disorders consented to participate in the study. Subjects were screened and demonstrated normative bladder emptying before being imaged while supine and standing using a sagittal curved linear array 2 to 5 MHz transducer over the suprapubic region. After pelvic floor muscle contraction was explained 4 parameters were measured 3 times each, including the direction of movement/displacement from freeze-frame ultrasound images, and endurance and coordination from ultrasound movie loops. The methodology for digitizing movie data were developed, tested and found to be reliable. New variables of endurance as a percent of maximum coordination amplitude and coordination as the amplitude between maximum and minimum effort were created. RESULTS: Overall 66% and 71% of subjects demonstrated anterior displacement of the pelvic floor during voluntary contraction while lying and standing, respectively, with no significant difference in lying vs standing. However, coordination displacement was greater while lying than standing. During 20-second contractions pelvic floor muscle activity attained peak amplitude at 5.5 seconds, followed by a marked decay with 1 or more cycles of muscular re-recruitment. It was observed that fatigue led to repeat recruitment of the rectus and oblique abdominal muscles. CONCLUSIONS: In children free of voiding dysfunction pelvic floor displacement and coordination are highly variable. Noninvasive ultrasound of the pelvic floor provided visual assessment of muscular activity, a biofeedback component for the patient and measurement potential for the therapist.
Asunto(s)
Contracción Muscular , Diafragma Pélvico/fisiología , Adolescente , Niño , Femenino , Humanos , Masculino , Diafragma Pélvico/diagnóstico por imagen , Diafragma Pélvico/fisiopatología , Postura , Valores de Referencia , Ultrasonografía , Incontinencia Urinaria/diagnóstico por imagen , Incontinencia Urinaria/fisiopatologíaRESUMEN
PURPOSE: Constipation in children increases the likelihood of urinary incontinence, bladder overactivity, dyscoordinated voiding, a large capacity, poorly emptying bladder, recurrent urinary tract infection and deterioration of vesicoureteral reflux. We present a consensus related to the assessment, diagnosis and treatment of children with bowel dysfunction coexisting with a known disorder of urinary continence or voiding coordination. MATERIALS AND METHODS: A panel of international multidisciplinary clinicians working on pediatric continence care was invited to participate in the First International Children's Continence Society Bowel Dysfunction Workshop. The seminar sought to address the interrelationship of bowel dysfunction with disorders of urinary continence or voiding mechanics. RESULTS: Constipation is an end point defined by a constellation of symptoms, including infrequent passage of stool, difficulty passing stool, feces that are either large and hard or in small pieces, abdominal pain, palpable stool in the abdomen, stool in the rectal vault, loading on x-ray or fecal soiling. Assessment was done to identify potential organic causes of constipation, clarify symptoms, and identify altered motor behavior and abdomino/pelvic floor muscle incoordination. Whether the underlying problem was one of stool consistency, poor cognition, motivation or fear on the part of the child, or whether it related to gut motility, rectal sensation, stool retention or disordered emptying mechanics, the definitive therapy begins with rectal emptying of impacted stool followed by maintenance of regular soft stools to eliminate fear of pain with defecation. CONCLUSIONS: Constipation is a challenge to the clinician but with comprehensive assessment and systematic intervention children can achieve independent bowel emptying, which positively impacts bladder function.
Asunto(s)
Estreñimiento/complicaciones , Niño , Estreñimiento/diagnóstico , Estreñimiento/fisiopatología , Estreñimiento/terapia , HumanosRESUMEN
In Saccharomyces cerevisiae, inactivation of the nuclear gene YME1 causes several phenotypes associated with impairment of mitochondrial function. In addition to deficiencies in mitochondrial compartment integrity and respiratory growth, yme1 mutants grow extremely slowly in the absence of mitochondrial DNA. We have identified two genetic loci that, when mutated, act as dominant suppressors of the slow-growth phenotype of yme1 strains lacking mitochondrial DNA. These mutations only suppressed the slow-growth phenotype of yme1 strains lacking mitochondrial DNA and had no effect on other phenotypes associated with yme1 mutations. One allele of one linkage group had a collateral respiratory deficient phenotype that allowed the isolation of the wild-type gene. This suppressing mutation was in ATP3, a gene that encodes the gamma subunit of the mitochondrial ATP synthase. Recovery of two of the suppressing ATP3 alleles and subsequent sequence analysis placed the suppressing mutations at strictly conserved residues near the C terminus of Atp3p. Deletion of the ATP3 genomic locus resulted in an inability to utilize nonfermentable carbon sources. atp3 deletion strains lacking mitochondrial DNA grew slowly on glucose media but were not as compromised for growth as yme1 yeast lacking mitochondrial DNA.
Asunto(s)
ADN Mitocondrial/genética , ATPasas de Translocación de Protón/genética , Saccharomyces cerevisiae/genética , Supresión Genética , Secuencia de Aminoácidos , Escherichia coli/genética , Datos de Secuencia Molecular , Fenotipo , Plásmidos , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/crecimiento & desarrollo , Alineación de SecuenciaRESUMEN
The DNA binding domain of the single-stranded DNA binding protein from Escherichia coli has been overproduced, purified and crystallized in a form suitable for X-ray diffraction studies. Crystals were produced by dialysis against low ionic strength buffer at high pH. The crystals belong to space group I222 or I2(1)2(1)2(1) with a = 82.47 A, b = 65.27 A and c = 46.50 A. Data were collected at several temperatures and a significant improvement in data quality was observed with a decrease in temperature. On occasion, with the decrease in temperature, a transformation to a primitive space group was observed and temperature appears to play a key role in this transformation. A complete native data set has been collected to 2.57 A at -15 degrees C.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Escherichia coli/química , Secuencia de Bases , Sitios de Unión , Frío , Cristalografía por Rayos X , ADN de Cadena Simple , Datos de Secuencia Molecular , OligodesoxirribonucleótidosRESUMEN
Coliphage N4 is a double-stranded DNA virus that requires the sequential activity of three different RNA polymerases during infection. The N4 virion RNA polymerase, which is carried in the virion and is injected with the DNA at the start of infection, is responsible for the synthesis of N4 early RNAs. In vitro, the virion RNA polymerase can transcribe double-stranded N4 DNA accurately and efficiently but only when the DNA is denatured. We have shown previously that the activity of DNA gyrase is required for in vivo early N4 transcription. We report here that Escherichia coli single-stranded DNA-binding protein (SSB) is also required for N4 early transcription. In vitro, linear or relaxed templates cannot be activated by SSB; however, supercoiled template and SSB allow the virion polymerase to recognize its promoters on duplex DNA and activate transcription. The effects of supercoiling are limited to transcript initiation and are not required for transcript elongation. The activation is specific for SSB; no other single-stranded DNA-binding proteins can substitute. Therefore, SSB is one of a small number of proteins that function to stimulate both replication and transcription. The basis for the specificity of SSB, the mechanism of transcriptional activation by SSB and template supercoiling, and their role in the N4 transcriptional program during development are discussed.
Asunto(s)
Colifagos/enzimología , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/metabolismo , Transactivadores/metabolismo , Secuencia de Bases , ADN Superhelicoidal/metabolismo , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , ARN/genética , Moldes Genéticos , Transactivadores/químicaRESUMEN
An endo-exonuclease (designated nuclease III) has been purified to near homogeneity from adult flies of Drosophila melanogaster. The enzyme degrades single- and double-stranded DNA and RNA. It has a sedimentation co-efficient of 3.1S and a strokes radius of 27A The native form of the purified enzyme appears to be a monomer of 33,600 dalton. It has a pH optimum of 7-8.5 and requires Mg2+ or Mn2+ but not Ca2+ or Co2+ for its activity. The enzyme activity on double-stranded DNA was inhibited 50% by 30 mM NaCl, while its activity on single-stranded DNA required 100 mM NaCl for 50% inhibition. Under the latter conditions, its activity on double-stranded DNA was inhibited approximately 98%. The enzyme degrades DNA to complete acid soluble products which are a mixture of mono- and oligonucleotides with 5'-P and 3'-OH termini. Supercoiled DNA was converted by the enzyme to nicked and subsequently to linear forms in a stepwise fashion under the condition in which the enzyme works optimally on single-stranded DNA. The amino acid composition and amino acid sequencing of tryptic peptides from purified nuclease III is also reported.
Asunto(s)
Drosophila melanogaster/enzimología , Endonucleasas/metabolismo , Exonucleasas/metabolismo , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Cromatografía DEAE-Celulosa , Cromatografía Líquida de Alta Presión , ADN de Cadena Simple , Electroforesis en Gel de Poliacrilamida , Endonucleasas/aislamiento & purificación , Exonucleasas/aislamiento & purificación , Calor , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Peso Molecular , Cloruro de Sodio , Especificidad por Sustrato , UltracentrifugaciónRESUMEN
The DNA and protein sequences of single-stranded DNA binding proteins (SSBs) encoded by the plP71a, plP231a, and R64 conjugative plasmids have been determined and compared to Escherichia coli SSB and the SSB encoded by F-plasmid. Although the amino acid sequences of all of these proteins are highly conserved within the NH2-terminal two-thirds of the protein, they diverge in the COOH-terminal third region. A number of amino acid residues which have previously been implicated as being either directly or indirectly involved in DNA binding are conserved in all of these SSBs. These residues include Trp-40, Trp-54, Trp-88, His-55, and Phe-60. On the basis of these sequence comparisons and DNA binding studies, a role for Tyr-70 in DNA binding is suggested for the first time. Although the COOH-terminal third of these proteins diverges more than their NH2-terminal regions, the COOH-terminal five amino acid residues of all five of these proteins are identical. In addition, all of these proteins share the characteristic property of having a protease resistant, NH2-terminal core and an acidic COOH-terminal region. Despite the high degree of sequence homology among the plasmid SSB proteins, the F-plasmid SSB appears unique in that it was the only SSB tested that neither bound well to poly(dA) nor was able to stimulate DNA polymerase III holoenzyme elongation rates. Poly [d(A-T)] melting studies suggest that at least three of the plasmid encoded SSBs are better helix-destabilizing proteins than is the E. coli SSB protein.
Asunto(s)
Conjugación Genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/química , Plásmidos , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , ADN Polimerasa III/metabolismo , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Datos de Secuencia Molecular , Poli A/metabolismo , Homología de Secuencia de Ácido Nucleico , TemperaturaRESUMEN
When E. coli single-stranded DNA binding protein (SSB) coats single-stranded DNA (ssDNA) in the presence of 1 mM MgCl2 it inhibits the subsequent binding of recA protein, whereas SSB binding to ssDNA in 12 mM MgCl2 promotes the binding of recA protein. These two conditions correspond respectively to those which produce 'smooth' and 'beaded' forms of ssDNA-SSB filaments. By gel filtration and immunoprecipitation we observed active nucleoprotein filaments of recA protein and SSB on ssDNA that contained on average 1 monomer of recA protein per 4 nucleotides and 1 monomer of SSB per 20-22 nucleotides. Filaments in such a mixture, when digested with micrococcal nuclease produced a regular repeating pattern, approximately every 70-80 nucleotides, that differed from the pattern observed when only recA protein was bound to the ssDNA. We conclude that the beaded ssDNA-SSB nucleoprotein filament readily binds recA protein and forms an intermediate that is active in the formation of joint molecules and can retain substantially all of the SSB that was originally bound.
Asunto(s)
ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Nucleoproteínas/metabolismo , Rec A Recombinasas/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Cromatografía en Gel , Cinética , Cloruro de Magnesio/farmacología , Nucleasa Microcócica/metabolismo , Pruebas de PrecipitinaRESUMEN
The adenovirus single-stranded DNA (ssDNA)-binding protein (DBP) is necessary for the elongation step in viral DNA replication. In an attempt to characterize the putative ssDNA-binding domain of the DBP, we purified and characterized the Ad2ts111A DBP, which contains a glycine-to-valine substitution at amino acid 280. This mutation is adjacent to that in the previously studied Ad2+ND1ts23. Ad2+ND1ts23 exhibits a temperature-sensitive defect in DNA replication, and its DBP has previously been shown to bind ssDNA with reduced affinity. Ad2ts111A DBP, like Ad2+ND1ts23, does not support adenovirus DNA replication in vitro at elevated temperatures. However, the Ad2ts111A DBP binds ssDNA more tightly than does Ad2+ND1ts23 and is not temperature sensitive in this function. To determine the nucleic acid-binding properties of DBP, we applied spectrofluorometric techniques, which had not been used previously to study adenovirus DBP. Using the homopolynucleotide poly(1,N6)-ethenoadenylic acid (poly(r epsilon A], we have determined that the binding site size is approximately 16 nucleotides. In 20 mM NaCl, the Ad2wt, Ad2ts111A, and Ad2+ND1ts23 DBP proteins all bound stoichiometrically to poly(r epsilon A) with overall apparent affinities above 108 M-1. Based on titrations carried out at higher salt concentrations, however, the stability of these complexes did appear to increase in the order Ad2+ND1ts23 less than Ad2ts111A less than Ad2wt. By these techniques, we have confirmed also that the DBP of another temperature-sensitive mutant, H5ts107, like the Ad2ts111A DBP, retains its ability to bind ssDNA even at a restrictive temperature utilizing the salt concentration compatible with adenovirus DNA replication in vitro. The H5ts107 DBP, which contains an amino acid substitution at position 413, is defective for in vitro replication at nonpermissive temperature but is not temperature sensitive for binding to ssDNA. In summary, our results indicate that the replication defects of the Ad2ts111A are similar to those of H5ts107 and cannot be attributed to defective, nonspecific ssDNA binding by the DBP. It appears that ssDNA binding by itself is not sufficient to account for the role of DBP in adenovirus DNA replication.
Asunto(s)
Proteínas de Unión al ADN/aislamiento & purificación , Ácidos Nucleicos/metabolismo , Adenoviridae/genética , Adenoviridae/fisiología , Sitios de Unión , Quimotripsina/metabolismo , Replicación del ADN , ADN de Cadena Simple/metabolismo , ADN Viral/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Mutación , Concentración Osmolar , Poli A/metabolismo , Polidesoxirribonucleótidos , Cloruro de Sodio/farmacología , Espectrometría de Fluorescencia , Temperatura , Moldes Genéticos , Replicación ViralRESUMEN
The complexes of point-mutated Escherichia coli single-stranded DNA-binding protein (Eco SSB) with poly-(2-thiouridylic acid) (poly S2U) have been studied by optical detection of magnetic resonance spectroscopy (ODMR). Previous work has determined that two of four tryptophan (Trp) residues in Eco SSB undergo stacking interactions with nucleic acid bases. Selective photoexcitation of S2U bases was performed and subsequent triplet----triplet energy transfer from S2U to nearby Trp residues in the protein took place. The zero-field splitting (ZFS) parameters and sublevel kinetics were determined for each Trp residue sensitized by S2U. The sublevel lifetimes of the two sensitized residues are similar to those of normal Trp. The ZFS parameters, on the other hand, show a dramatic reduction relative to those of the uncomplexed protein, implying a more polarizable environment for the sensitized Trp residues and/or charge transfer interactions with the S2U bases.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Escherichia coli/análisis , Poli U/metabolismo , Tionucleótidos/metabolismo , Transferencia de Energía , Cinética , Luz , Luminiscencia , Espectroscopía de Resonancia Magnética , TriptófanoRESUMEN
Complexes of point-mutated E. coli single-stranded DNA-binding protein (Eco SSB) with homopolynucleotides have been investigated by optical detection of magnetic resonance (ODMR) of the triplet state of tryptophan (Trp) residues. Investigation of the individual sublevel kinetics of the lowest triplet state of Trp residues 40 and 54 in the poly (dT) complex of Eco SSB-W88F,W135F (a mutant protein whose Trp residues at positions 88 and 135 have been substituted by Phe) shows that Trp 54 is the most affected residue upon stacking with thymine bases, confirming previous results based on SSB mutants having single Trp----Phe substitutions. (Zang, L. H., A. H. Maki, J. B. Murphy, and J. W. Chase. 1987. Biophys. J. 52:867-872). The Tx sublevel of Trp 54 shows a fourfold increase in the decay rate constant, as well as an increase in its populating rate constant by selective spin-orbit coupling. The two nonradiative sublevels show no change in lifetime, relative to unstacked Trp. For Trp 40, a weaker perturbation of Tx by thymine results in a sublevel lifetime about one-half that of normal Trp. Trp54 displays a 2[E]transition of negative polarity in the double mutant SSB complex with Poly (dT), but gives a vanishingly weak [D] - [E] signal, thus implying that the steady-state sublevel populations of Tx and Tz are nearly equal in this residue. Poly (5-BrU) induces the largest red-shift of the Eco SSB-W88F,W135F Trp phosphorescence 0,0-band of all polynucleotides investigated. Its phosphorescence decay fits well to two exponential components of 1.02 and 0.12 s, with no contribution from long-lived Trp residues. This behavior provides convincing evidence that both Trp 40 and 54 are perturbed by stacking with brominated uridine. The observed decrease in the Trp [D] values further confirms the stacking of the Trp residues with 5-BrU. Wave-length-selected ODMR experiments conducted on the [D[ + [E] transition of Eco SSB-W88F,W135F complexed with poly(5HgU) indicate the presence of multiple heavy atom-perturbed sites. Measurements made on poly (5-HgU) which each of its 4 Trp residues has been replaced in turn by Phe demonstrate that Trp 40 and 54 are the only Trp residues undergoing stacking with nucleotide bases, as previously proposed.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Polinucleótidos/metabolismo , Triptófano , Cinética , Poli T/metabolismoRESUMEN
In an extension of earlier studies on the Escherichia coli plasmid-encoded single-stranded DNA-binding proteins pIP71a SSB, F SSB and R64 SSB [Khamis, M. I., Casas-Finet, J. R., Maki, A. H., Ruvolo, P. P. & Chase, J. W. (1987) Biochemistry 26, 3347-3354; Casas-Finet, J. R., Khamis, M. I., Maki, A. H., Ruvolo, P. P. & Chase, J. W. (1987) J. Biol. Chem. 262, 8574-8593], we have investigated the binding of pIP231a SSB to natural and heavy-atom-derivatized single-stranded homopolynucleotides. Fluorimetric equilibrium binding isotherms indicate that pIP231a SSB has a greater solubility at low ionic strength than any other plasmid SSB protein investigated. Furthermore, its complex with mercurated poly(uridylic acid) [poly(Hg5U)] shows a greater resistance to disruption by salt than the other plasmid SSB complexes. Essentially complete binding of pIP231a SSB to poly(Hg5U) could be achieved, and time-resolved optically detected triplet-state magnetic resonance (ODMR) techniques could be applied to the complex. These methods allowed complete resolution of the three Trp chromophores of pIP231a SSB. Comparison of wavelength-selected ODMR results with those obtained for the poly(Hg5U) complex of a point-mutated chromosomal ssb gene product (Eco SSB) carrying substitutions of Phe for Trp [Khamis, M. I., Casas-Finet, J. R., Maki, A. H., Murphy, J. B. & Chase, J. W. (1987) J. Biol. Chem. 262, 10938-10945] confirm that Trp40 and Trp54 of pIP231a SSB are stacked in the complex, while Trp88 is not. This is the same distribution of stacked Trp residues found in Eco SSB. These results are confirmed further by specific effects observed on the ODMR signals of pIP231a SSB upon binding to poly(Br5U) and poly(dT), which are known to be caused by the stacking of Trp54 with nucleic acid bases.
Asunto(s)
Proteínas Bacterianas/genética , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Genes Bacterianos , Triptófano/genética , Sitios de Unión , ADN de Cadena Simple/análisis , Proteínas de Unión al ADN/análisis , Escherichia coli/análisis , Espectroscopía de Resonancia Magnética , Plásmidos , Polinucleótidos/farmacología , Solubilidad , Espectrometría de Fluorescencia , Triptófano/análisisRESUMEN
In a companion study, a number of P element insertions into the singed locus were characterized. Here is reported a detailed analysis of the structure and mutability of another P element insertion at sn, known as sncm. Under conditions which mobilize P elements, sncm mutates at high frequency to both wild-type (sn+) and to a much more extreme allele (snext). Wild-type revertants appear to represent precise or nearly precise excisions of the P element. Certainly two, and most likely all five, of the snext alleles studied result from the insertion of a duplicate copy of this P element into a nearby site in an inverted orientation. We propose a model in which both the sn+ and snext mutational events can be explained by excision of the P element from one chromatid followed by reintegration into the sister chromatid at a nearby site (intracistronic transposition). Finally, it is shown that the snext alleles are themselves unstable and the structure of a resulting chromosome aberration is examined.
Asunto(s)
Mapeo Cromosómico , Elementos Transponibles de ADN , Drosophila melanogaster/genética , Alelos , Animales , Secuencia de Bases , ADN/aislamiento & purificación , Genes , Inmunoquímica , Microscopía Electrónica de Rastreo , MutaciónRESUMEN
The individual sublevel kinetics of the lowest triplet state of tryptophan 54 (Trp 54) which is highly perturbed in the complex of Escherichia coli single-stranded DNA binding protein (Eco SSB) with poly(deoxythymidylic) acid (poly[dT]) have been studied by optically detected magnetic resonance (ODMR) spectroscopy. The triplet sublevel decay constants of Trp 54, kx, ky, kz, are 0.99, 0.072, and 0.045 s-1, respectively, in the poly(dT) complex of a point-mutated Eco SSB in which Trp 88 is substituted by phenylalanine. Tx is the only radiative triplet sublevel. Negative polarity of the Tx----Tz and Tx----Ty phosphorescence-detected ODMR signals results from the steady state population pattern, nx greater than ny, nz, and implies that the relations, px greater than or equal to 14py, and px greater than or equal to 22pz exist for the relative populating rates. Spin-orbit coupling between radiative singlet states and the Tx sublevel of the lowest triplet state of Trp 54 is enhanced selectively upon complexing of Eco SSB with poly(dT).
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Poli T/metabolismo , Polidesoxirribonucleótidos/metabolismo , Triptófano , Cinética , Unión ProteicaRESUMEN
DNA ligase has been purified to homogeneity from 6-12 h Drosophila melanogaster embryos (Rabin, B. A., Hawley, R. S., and Chase, J. W. (1986) J. Biol. Chem. 261, 10637-10645). This enzyme had an apparent Km for ATP of 1.6 microM. Of a variety of nucleotides tested, only adenosine 5'-O-(3-thio)triphosphate could substitute for ATP in the joining reaction. The enzyme was competitively inhibited by dATP, with an apparent Ki of 2.3 microM. The apparent Km for DNA using p(dT)20 annealed with poly(dA) as substrate was 1.0 microM. Studies utilizing synthetic homopolymers showed that in addition to joining DNA to DNA, this enzyme could join the 5'-phosphoryl termini of RNA to the 3'-hydroxyl termini of DNA or RNA, when they were annealed with DNA. In addition, p(dT)7U could be joined when annealed with poly(dA). No joining was detected when RNA served as the template. Drosophila DNA ligase also catalyzed the joining of oligonucleotides containing a single mismatched nucleotide at their 3'-hydroxyl termini, as well as DNA containing short, complementary 5'-protruding ends, and in the presence of polyethylene glycol 6000, blunt-ended duplex DNA. The overall reaction mechanism was shown to be identical to that of the homologous prokaryotic DNA ligases. The joining reactions catalyzed by the Drosophila and T4 DNA ligases were shown to be reversible. Incubation of superhelical closed circular DNA molecules with the purified enzymes and AMP resulted in the production of a population of DNA molecules which had lost most, if not all, of their superhelical density.
Asunto(s)
ADN Ligasas/metabolismo , Drosophila melanogaster/enzimología , Polinucleótido Ligasas/metabolismo , Animales , Drosophila melanogaster/embriología , Embrión no Mamífero/enzimología , Cinética , Especificidad por Sustrato , TermodinámicaRESUMEN
The binding of both wild-type and point-mutated E. coli single-stranded DNA-binding (SSB) protein to poly(deoxythymidylic acid) has been studied by fluorescence and optical detection of triplet state magnetic resonance spectroscopy. Involvement of tryptophan residues 40 and 54 in stacking interactions with nucleotide bases has been inferred earlier from such studies. Investigation of a point mutation in the E. coli SSB gene product obtained by site specific oligonucleotide mutagenesis in which Phe-60 is replaced by alanine strongly suggests the participation of Phe-60 in the binding process, possibly by the formation of an extended stacking structure by Trp-54, thymine and Phe-60. This hypothesis is supported by results on the point mutations in which His-55 is replaced by either leucine or tyrosine.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Escherichia coli , Fenilalanina , Poli T/metabolismo , Espectrometría de Fluorescencia , Relación Estructura-Actividad , TriptófanoRESUMEN
To relate the roles of Escherichia coli SSB in recombination in vivo and in vitro, we have studied the mutant proteins SSB-1 and SSB-113, the variant SSBc produced by chymotryptic cleavage, the partially homologous variant F SSB (encoded by the E. coli sex factor), and the protein encoded by gene 32 of bacteriophage T4. All of these, with the exception of SSB-1, augmented both the initial rate of homologous pairing and strand exchange promoted by RecA protein. From these and related observations, we conclude that SSB stimulates the initial formation of joint molecules by nonspecifically promoting the binding of RecA protein to single-stranded DNA; that SSB plays no role in synapsis of the RecA nucleoprotein filament with duplex DNA; that stimulation of strand exchange by SSB is similarly nonspecific; and that all members of the class of proteins represented by SSB, F SSB, and gene 32 protein may play equivalent roles in making single-stranded DNA more accessible to RecA protein.
Asunto(s)
ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/farmacología , Rec A Recombinasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Mutación , Fagos T/genética , Fagos T/metabolismoRESUMEN
Fluorescence and optical detection of triplet state magnetic resonance (ODMR) spectroscopy have been employed to study the complexes formed between single-stranded polynucleotides and Escherichia coli ssb gene products (SSB) in which tryptophans 40, 54, and 88 are selectively, one residue at a time, replaced by phenylalanine using site-specific oligonucleotide mutagenesis. Fluorescence titrations and ODMR results indicate that tryptophans 40 and 54 are the only tryptophan residues in E. coli single-stranded DNA binding protein that are involved in stabilizing the protein-nucleic acid complexes via stacking interactions. Wavelength-selected ODMR measurements on E. coli SSB reveal the presence of two spectrally distinct tryptophan sites (Khamis, M. I., Casas-Finet, J. R., and Maki, A. H. (1987) J. Biol. Chem. 262, 1725-1733). Our present results indicate that tryptophan 54 belongs to the blue-shifted site, while tryptophan 40 belongs to the red-shifted site of the protein.
Asunto(s)
ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/análisis , Polinucleótidos/metabolismo , Triptófano , Secuencia de Aminoácidos , Sitios de Unión , Proteínas de Unión al ADN/genética , Espectroscopía de Resonancia Magnética , Mutación , Poli T/metabolismo , Poli U/análogos & derivados , Poli U/metabolismo , Espectrometría de FluorescenciaRESUMEN
Optically detected magnetic resonance (ODMR) methods were employed to study three single-stranded DNA binding (SSB) proteins encoded by plasmids of enteric bacteria: pIP71a, R64, and F. Equilibrium binding isotherms obtained by fluorescence titrations reveal that the complexes of the plasmid SSB proteins with heavy atom modified polynucleotides are readily disrupted by salt. Since all the plasmid SSB proteins show limited solubility at low ionic strength (pIP71a greater than R64 greater than F), we were able to bind only the pIP71a protein to mercurated poly(uridylic acid) [poly(5-HgU)] and brominated poly(uridylic acid) [poly(5-BrU)]. ODMR results reveal the existence of at least one heavy atom perturbed, red-shifted, stacked Trp residue in these complexes. Amplitude-modulated phosphorescence microwave double resonance spectra display selectively the phosphorescence associated with Hg-perturbed Trp residue(s) in the pIP71a SSB protein-poly(5-HgU) complex, which has a broad, red-shifted 0,0-band. Our results suggest that Trp-135 in Escherichia coli SSB, which is absent in the plasmid-encoded SSB proteins, is located in a polar environment and is not involved in stacking interactions with the nucleotide bases. Phosphorescence spectra and lifetime measurements of the pIP71a SSB protein-poly (5-BrU) complex show that at least one Trp residue in the complex does not undergo stacking. This sets a higher limit of two stacking interactions of Trp residues with nucleotide bases in complexes of pIP71a SSB with single-stranded polynucleotides.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Poli U/análogos & derivados , Escherichia coli/metabolismo , Cinética , Mediciones Luminiscentes , Espectroscopía de Resonancia Magnética/métodos , Concentración Osmolar , Plásmidos , Poli U/metabolismo , TriptófanoRESUMEN
Optically detected magnetic resonance (ODMR) spectroscopy has been applied to several single-stranded DNA-binding (SSB) proteins encoded by conjugative plasmids of enteric bacteria. Fluorimetric equilibrium binding isotherms confirm their preferential binding to single-stranded DNA and polynucleotides and reveal a limited protein solubility at low ionic strength. The plasmid SSB-like proteins show the highest affinity for polydeoxythymidylic acid; these complexes are the least sensitive to disruption by salt. ODMR data on these complexes suggest the existence of stacking interactions between tryptophan residue(s) and thymine bases, as evidenced by spectral red shifts of the tryptophan phosphorescence 0,0 band, reduction of the magnitude of D zero field splitting parameter, and a dramatic reversal of the polarity of the ODMR signals. Wavelength-selected ODMR results point to the existence of two distinct tryptophan sites in these complexes. The triplet state properties of the red-shifted site are drastically altered by its interaction with the thymine bases. The chromosomal Escherichia coli SSB protein-poly(dT) complex shows an additional tryptophan site with zero field splitting parameters similar to those of the free protein. This site can be attributed to Trp-135, which is missing in each of the other plasmid SSB proteins, suggesting that this particular residue is not involved in the interaction with polynucleotides.